Synthesis of Flavonols and Assessment of Their Biological Activity as Anticancer Agents

A series of flavanols were synthesized to assess their biological activity against human non-small cell lung cancer cells (A549). Among the sixteen synthesized compounds, it was observed that compounds 6k (3.14 ± 0.29 µM) and 6l (0.46 ± 0.02 µM) exhibited higher potency compared to 5-fluorouracil (5-Fu, 4.98 ± 0.41 µM), a clinical anticancer drug which was used as a positive control. Moreover, compound 6l (4’-bromoflavonol) markedly induced apoptosis of A549 cells through the mitochondrial- and caspase-3-dependent pathways. Consequently, compound 6l might be developed as a candidate for treating or preventing lung cancer.


Introduction
Flavonoids are naturally occurring polyphenolic compounds.More than 10,000 flavonoids have been detected and categorized into subclasses [1].They are isolated from a wide range of plant families and species, and exhibit certain pharmacological activities such as antioxidant [2], anti-inflammatory [3,4], antimicrobial [5,6], antiallergenic [7], anticancer [8,9] and antiviral [10].Flavonoids are natural antioxidants since they possess a reactive oxygen species (ROS) that will damage the membranes and DNA in mammals [11].The various classes of flavonoids differ in the level of oxidation and pattern of substitution on the C ring (Figure 1).The double bond between C2-C3 and the oxo group at C4 of C ring, and the position of the B ring are crucial determinants for their anticancer activity [12].Flavonoids act by multiple mechanisms but further studies on target selectivity and specificity of flavonoids are necessary to establish them as anticancer therapeutics [12].The most studied flavonols, a class of flavonoids, are quercetin, kaempferol, galangin, and myricetin, widely present in fruits, vegetables, tea, cocoa, and red wine (Figure 2) [13].In addition, previous research results indicate the inhibitory effects of flavonoids such as apigenin and luteolin as well as the flavonol quercetin and its derivatives on various leukemia cell lines [14].These natural compounds can be prototypes for broad-spectrum chemotherapy drugs [14].Flavonoids have been reported to have an excellent safety profile (no toxicity at up to 140 g/day), with no known significant adverse effects [15].Pietta et al. reported that the 3-OH group in the C ring is essential to generate a high radicalscavenging activity [16].Additionally, antioxidants help human beings reduce cancer risks [17].Furthermore, this activity is enhanced when an additional hydroxyl group, such as myricetin, is present on the B ring [16].Research reports that quercetin (Figure 2) has multiple pharmacological properties, including neuroprotective [18], anticancer [19][20][21][22], and antiviral [23,24] properties.
Molecules 2024, 29, 2041 2 of 11 radical-scavenging activity [16].Additionally, antioxidants help human beings reduce cancer risks [17].Furthermore, this activity is enhanced when an additional hydroxyl group, such as myricetin, is present on the B ring [16].Research reports that quercetin (Figure 2) has multiple pharmacological properties, including neuroprotective [18], anticancer [19][20][21][22], and antiviral [23,24] properties.Natural and synthetic flavonoids have been developed as agents against non-small lung cancer [19,25].Previously, we reported the synthesis of halo-substituted chalcones and azachalcones to inhibit the pro-inflammatory response [26].Since flavonols can be synthesized from chalcones, we aim to explore the potential of halo-substituted flavonols.radical-scavenging activity [16].Additionally, antioxidants help human beings reduce cancer risks [17].Furthermore, this activity is enhanced when an additional hydroxyl group, such as myricetin, is present on the B ring [16].Research reports that quercetin (Figure 2) has multiple pharmacological properties, including neuroprotective [18], anticancer [19][20][21][22], and antiviral [23,24] properties.Natural and synthetic flavonoids have been developed as agents against non-small lung cancer [19,25].Previously, we reported the synthesis of halo-substituted chalcones and azachalcones to inhibit the pro-inflammatory response [26].Since flavonols can be synthesized from chalcones, we aim to explore the potential of halo-substituted flavonols.Natural and synthetic flavonoids have been developed as agents against non-small lung cancer [19,25].Previously, we reported the synthesis of halo-substituted chalcones and azachalcones to inhibit the pro-inflammatory response [26].Since flavonols can be synthesized from chalcones, we aim to explore the potential of halo-substituted flavonols.Therefore, in this study, we investigated the activity of sixteen synthesized flavonols against human non-small lung cancer cells (A549).
Molecules 2024, 29, 2041 3 of 11 Therefore, in this study, we investigated the activity of sixteen synthesized flavonols against human non-small lung cancer cells (A549).
The effect of treating the A549 cells with compound 6l (20 µM) on the expression of apoptosis-related proteins was investigated (Figure 3).The results revealed a decrease in the expression level of the anti-apoptotic protein Bcl-2, while that of the pro-apoptotic protein Bax increased.Caspase-3 activation is a hallmark of apoptosis.Thus, compound 6l increases the expression level of cleaved caspase-3 (active caspase-3).The results showed that compound 6l induced apoptosis in A549 cells through mitochondrial-and caspase-3-dependant pathways.

General Procedures
All chemicals were purchased from either Acros or Alfa from Uniworld in Taiwan.The 1 H-and C 13 -NMR data were recorded on a Bruker 600 MHz Ultrashield instrument (Bruker, Billerica, MA, USA).The chemicals were reported in parts per million (ppm) relative to the residual solvent ( 1 H for DMSO-d 6 : 2.49 ppm; 13 C NMR for DMSO-d 6 : 39.7 ppm).The reaction progress was monitored by thin-layer chromatography (Analtech Silica gel HLF UV254, Analtech Inc. Newark, DE, USA) and stained with either KMnO 4 or p-anisaldehyde solutions.The melting points were determined by an open capillary tube on an MP-2 apparatus.The molecular weights of compounds were determined by a Thermo LCQ Fleet ion trap mass spectrometer.

Mass Determination
Using a micropipette, the sample (1 µL) was loaded onto a graphite paper cut into a triangle (10 × 10 mm), washed with MeOH, and cleaned with Kimwipe paper.An ion trap mass spectrometer (LCQ Duo, Finnigan, San Jose, CA, USA) equipped with a paper-spray ionization (PSI) source was employed to determine the molecular weights of the samples.A high voltage (3.5 kV) was applied for sample ionization.The MS spectra scans were collected in the positive and negative ion modes in the m/z range of 100-400.

General Preparation of Chalcones
NaOH (50%, 3.0 equiv.) was added to a solution of acetophenone (1.0 equiv.) in EtOH (0.2 M) and stirred at ambient temperature for 30 min.Subsequently, the corresponding aldehyde (1.2 equiv.) was added to the mixture in EtOH (0.2 M) at ambient temperature.The progress of the reaction was monitored by thin-layer chromatography (TLC) until the aldehyde was consumed.The mixture was acidified with HCl (2N) and added distilled water.The precipitate was filtered by suction filtration and washed with EtOH to obtain chalcones 4a-l and 5i-l.

General Preparation of Flavonols
A solution of the corresponding 2-hydroxyacetophenone (1.0 equiv.)and NaOH (50%, 5.0 equiv.) in MeOH was added to H 2 O 2 (35%).The mixture was stirred in an ice bath and TLC monitored the progress of the reaction.At the end of the reaction, the mixture was acidified with HCl (2N), and distilled water was added to allow a precipitate formation.The precipitate was washed with cold MeOH to obtain flavonols 6i-l and 7i-l.

In Vitro Cytotoxicity Assay
Cell viability was evaluated using the MTT assay [35,36] to further assess cytotoxicity.The compound stock solution was stored in dimethyl sulfoxide (DMSO) at a concentration of 100 mM at −20 • C and thawed immediately before use.Briefly, the cells were incubated in 96-well culture plates (3 × 10 3 cells in 200 µL per well).After 24 h, cells were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 µM) of all 16 synthesized compounds, and 5-FU was used as the positive control.After 72 h, the attached cells were treated with an MTT reagent (0.5 mg/mL to 100 µL of each well) and incubated at 37 • C for 3 h.This reagent was then removed, DMSO (100 µL) was added to each well to dissolve the formazan metabolite, and the amount of formazan was quantified by measuring the absorbance at 570 nm using an ELISA plate reader (TECAN Spark, Tecan Group Ltd., Männedorf, Switzerland) (µ Quant).The optical density of formazan formed in the control (untreated) cells was 100% viability.

Western Blotting Analysis
Western blotting was performed according to a previously described method [35,36].Briefly, the cells were seeded in 6-well culture plates.After reaching 85-90% confluence, cells were treated with 6l (0.25, 0.5, 1, and 2 µM) and 5-FU (5 µM) followed by incubation for 48 h.The cells were then collected and lysed using a radioimmunoprecipitation assay (RIPA) buffer.Lysates of the total protein were separated on sodium dodecyl sulfate polyacrylamide gels (12.5%) and transferred to polyvinylidene difluoride membranes.The membranes were blocked with a bovine serum albumin (2%, BSA) solution and incubated with anti-Bax, anti-Bcl-2 (Cell Signaling Inc., Danvers, MA, USA), anti-caspase-3, and anti-β-actin (GeneTex Inc., Irvine, CA, USA) primary antibodies at 4 • C overnight.Each membrane was washed three times with Tris-buffered saline containing Tween 20 (0.1%, TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h.Finally, the membranes were developed using an enhanced chemiluminescence (ECL) detection kit and visualized using an ImageQuant LAS 4000 Mini bio-molecular imager (GE Healthcare, Marlborough, MA, USA).Band densities were quantified using ImageJ software 1.53a (BioTechniques, New York, NY, USA).

Statistical Analysis
All results are presented as mean ± SEM.Statistical analysis was executed using Student's t-test.A probability of 0.05 or less was considered to be statistically significant.Microsoft Excel 2019 was used for the statistical and graphical assessment.All experiments were executed at least 3 times.

Conclusions
In conclusion, among the sixteen synthesized flavonoids, 2-(4-bromophenyl)-3-hydroxy-4H-chromen-4-one (6l) exhibited the highest cytotoxic activity against A549 cells.Furthermore, compounds 6a, 6j, and 6k exhibited activities comparable to 5-FU.The structure-activity relationship studies revealed that the halogenation at C'-4 in the B ring enhanced the cytotoxic effects against A549 cells.The Western blot analysis confirmed that compound 6l induced apoptosis in A549 cells via mitochondrial-and caspase-3-dependant pathways (Figure 4).The present study suggests that bioactive synthetic compounds 6a, 6j, 6k, and 6l (especially 6l), are potential cytotoxic agents and could be promising candidates for developing novel anticancer drugs.

Conclusions
In conclusion, among the sixteen synthesized flavonoids, 2-(4-bromophenyl)-3-hydroxy-4H-chromen-4-one (6l) exhibited the highest cytotoxic activity against A549 cells.Furthermore, compounds 6a, 6j, and 6k exhibited activities comparable to 5-FU.The structure-activity relationship studies revealed that the halogenation at C'-4 in the B ring enhanced the cytotoxic effects against A549 cells.The Western blot analysis confirmed that compound 6l induced apoptosis in A549 cells via mitochondrial-and caspase-3-dependant pathways (Figure 4).The present study suggests that bioactive synthetic compounds 6a, 6j, 6k, and 6l (especially 6l), are potential cytotoxic agents and could be promising candidates for developing novel anticancer drugs.

Figure 4 .
Figure 4. Schematic diagram for cancer cell apoptosis mechanism of compound 6l in A549 cells.

Table 1 .
Inhibitory effects of compounds against human non-small cell lung cancer cells (A549).

Table 1 .
Inhibitory effects of compounds against human non-small cell lung cancer cells (A549).